DESCRIPTION: (From the Applicant's proposal): Gerstmann-Straussler-Schienker syndrome (GSS) is an inherited human prion disease with spongiform degeneration, astrocylic gliosis, prion protein (PrP) amyloid plaques, and neurofibrillary tangles. Point mutations at condons 102, 105, 117, 145, 198 and 217 of the prion protein gene (PRNP) segregate with this disorder. Amyloid plaque cores include internal PrP fragments of 7, 11, 15-20, and 25-30 kDa that polymerize into insoluble fibrils. The subcellular site and mechanism of production of these fragments is not known. PI's initial studies of an in vitro transfected cell model of GSS subtype with the Q217R mutation show that the localization, processing, and fate of mutant PrP (PrPM) and its fragments differ from those of normal PrP (PrPC) in the following ways: a) About 50 percent of PrPC is normally truncated at a site that disrupts the amyloidogenic region, but much smaller amounts of PrPM are truncated, and a significant proportion is cleaved at a unique site that does not disrupt the amyloidogenic region. b) All three forms of full-length and truncated PrPC are expressed at the cell surface, but only one truncated form, and small amounts of full-length PrPM are at the surface. Thus, cleavage of PrPC and PrPM may occur in different subcellular compartments, or the cleaved fragments are routed differentially. It is therefore important to identify the subcellular site(s) of cleavage of PrP. c) Significant quantities of PrPC and PrPM are released extracellularly into the culture medium. Their differential processing by microglia may also contribute to the amyloidogenic process. d) A proportion of PrPM is synthesized as an abnormal, relatively stable isoform that does not leave the endoplasmic reticulum (ER). The mechanisms of its retention and eventual degradation are not clear. ER-resident chaperones may be involved, and compromise of the cellular quality-control with age may affect its accumulation, extent of intracellular aggregation, and eventual processing by microglia after cell-death. The proposed study will evaluate: a) the subcellular site(s) of cleavage of PrPC and PrPM, b) whether secreted PrP is processed extracellularly, c) which ER-chaperones associate with the abnormal isoform of PrPM, and d) the distribution of normal and mutant PrP and its fragments in polarized cells. The experimental approach involves: a) identification of site(s) of cleavage by blocking transport of PrP at different sites, b) characterization of secreted PrP and its processing by neuroblastoma cells or primary cultures of microglia, c) evaluation of polarized distribution of PrPC, PrPM, and their fragments in polarized CaCo2 cells, and c) co-immunoprecipitation to check for associated ER-resident chaperones.